The overall goal of this study is to investigate the molecular basis for the neuroendocrine disease familial neurohypophyseal diabetes insipidus (FNDI). FNDI is an inherited disorder caused by specific mutations in the gene that encodes pre-provasopressin. In this disease, mutant pro-vasopressin is misfolded and accumulates in the ER, is missorted to the constitutive secretory pathway and is not processed correctly. The hypothesis is that accumulation of pro-vasopressin mutations in the ER or Golgi due to improper folding or sorting causes cytotoxic effects that destroy the vasopressin producing cells. In order to test this hypothesis, a chimeric protein, pro- vasopressin-green fluorescent protein (VP-GFP) has been created that allows the mutants to be followed by fluorescent microscopy and immunoprecipitation. In Specific Aim 1 the intracellular fate of FNDI mutations to provasopressin will be studied. Ten FNDI-mutants will be expressed in Neuro-2a and AtT20 cells and their retention, processing and secretion analyzed by confocal and immuno-electron microscopy and pulse-chase experiments. Specific Aim 2 involves investigation of other proteins affected by the mutations, i.e., ER chaperones, proteasome enzymes, secretory granule proteins. In Specific Aim 3, the hypothesis that accumulation of the FNDI-mutations causes cytotoxicity will be tested. Two of the FNDI-mutations will be stably expressed and the cells evaluated to determine if the mutants are cytotoxic and the mechanism by which they produce the cytotoxicity, either necrosis or apoptosis or both. The fate of the mutant VP-GFP in all three specific aims will be followed by confocal microscopy, immunoelectron microscopy and pulse-chase secretion immunoprecipitation experiments either directly or with antibodies to GFP, AVP or neurophysin. Necrosis and apoptosis will be determined using specific assays, e.g., TUNEL, Comet, LDH and PS. The results from these experiments will contribute to understanding the molecular and cellular mechanisms that cause degeneration of vasopressin secreting cells in FNDI and possibly other neurodegenerative and ER storage diseases.